The mechanism of action of carcinostatic agents, such as BCNU and CCNU, will be investigated. These nitrosourea derivatives have been shown to inhibit DNA synthesis in vivo. In a preliminary study we observed that in vitro these agents inhibit one of two forms of purified non-mitochondrial DNA polymerases (I and II) from rat liver and hepatomas. DNA polymerase I, an enzyme that does not change with proliferation, is insensitive to these agents. However, DNA polymerase II is inhibited by these agents and it's activity correlates positively with the rate of proliferation. In normal proliferating and neoplastic tissues DNA polymerase II is also associated with other enzymes for DNA biosynthesis as a multi-enzyme complex. In this study we will determine in vitro the mechanism of inhibition of purified DNA polymerase II by these agents. This will include studies of: the essential substituents of the agents for inhibition, the kinetics and stoichiometry of the reaction(s) and the site(s) of binding of the agents to homogeneous DNA polymerase II. We will also investigate the effect of these agents on other enzymes of the multi-enzyme complex that includes: ribonucleotide reductase, thymidine kinase and thymidylate synthetase, in addition to DNA polymerase II. We will also investigate the effect of these agents on semi-conservative DNA synthesis in vitro with isolated nuclei from regenerating rat liver and hepatomas.